Varicella zoster virus (VZV) causes chickenpox (varicella) and shingles (herpes zoster). The understanding of VZV pathogenesis and the development of well-characterized vaccines have been complicated by difficulties in mutant viruses generation, and the lack of relevant in vitro cell culture systems. The broad objectives of the application are to introduce bacterial artificial chromosome (BAC) technology to VZV research, and to begin an in-depth analysis of the VZV ORF9 gene product. We hypothesize that the ORF9 protein is crucially involved in direct cell-to-cell spread but not virus egress via apical surfaces of cells of the epidermis in a skin organ culture system. Three specific aims are presented: Specific aim 1: Generate and mutagenize a VZV P-Oka BAC (pP-Oka). BAC technology shall be introduced to VZV research to facilitate the analysis of VZV genes. The VZV P-Oka BAC will be generated exactly as previously described for BACs of closely related Alphaherpesvirinae. After introduction of mini-F vector sequences into the intergenic region between ORF64 and ORF65 into the P-Oka genome by conventional homologous recombination, circular viral genomes will be transformed and maintained in Escherichia coli DH10B cells. Finally, ORF9-encoding sequences will be deleted and mutated in pP-Oka BAC by recombination in E. coli. Specific Aim 2: Test recombinant VZV reconstituted from BAC clones in cultured cells. MeWo cells, or MeWo cells that constitutively express the ORF9 protein (MeWo-9) will be transfected with the parental or mutagenized VZV BAC clones. Reconstituted viruses will be analyzed for their growth properties in these cells. The MeWo-9 cells will be generated because we hypothesize that the ORF9 product may be essential for direct cell-to-cell spread of VZV. Mutant viruses will be tested in a novel skin organ culture system, in which free infectious VZV is released after inoculation of infected MeWo cells. While we predict the ORF9 protein to be of great importance for direct cell-to-cell spread in cultured MeWo cells, virus egress in the skin organ culture system may be unaffected in its absence. This hypothesis is addressed by inoculation of the skin organ culture with MeWo-9 cells infected with the ORF9-negative viruses. Specific Aim 3: Test recombinant VZV in the SCID-hu mouse model. The growth properties of VZV generated from the cloned and/or mutated pP-Oka BAC will be tested in SCID-hu mice, a small animal model for VZV pathogenesis and the only model that allows the study of VZV replication in T cells in vivo [2-4]. The tropism of the recombinant viruses for both human skin and T cells will be tested. Human skin and/or conjoint thymus and liver tissue will be implanted into homozygous C.B-scid/beige mice, and infected MeWo or MeWo-9 cells will be used to inoculate the grafts. Virus replication in the implants will be examined by virus titrations, real-time PCR, histopathology and immunohistochemistry.